Development of a quantitative duplex droplet digital PCR for the detection of genetically modified COT102 cotton event

BACKGROUND: With the implementation of genetically modified (GM) component labeling thresholds and low-level presence requirements in an increasing number of countries and regions, the demand for accurate quantitative detection has grown substantially. Meanwhile, digital PCR exhibits significant advantages over conventional real-time PCR for the quantification of GM components. OBJECTIVE: In this study, a quantitative method of GM COT102 cotton event using duplex droplet digital PCR (ddPCR) was developed. METHODS: The duplex ddPCR system was designed to simultaneously quantify the copy number (CN) concentrations of the COT102 event-specific sequence and those of AdhC, an endogenous reference gene of cotton, within a single reaction. The ratio of the CN concentrations of COT102 to that of AdhC was calculated as the percentage of GM component. RESULTS: The relative limit of quantification (LOQrel.) for GM component COT102 was 0.1%, with the linear quantification ranges of COT102 and AdhC determined as 0.61-216 copies/μL and 0.54-261 copies/μL. CONCLUSION: A quantitative method using duplex ddPCR for the detection of GM COT102 cotton event was established. HIGHLIGHTS: This method is completely capable of meeting the quantification requirements worldwide, even the regions with the lowest labeling threshold, and is suitable for the detection of GM components in raw cotton, cottonseed oil and cottonseed meal.